The present invention relates to immunoparticles effective as diagnostic reagents for immunological tests and a process for preparing the same. More particularly, it is concerned with improved immunoparticles prepared by immobilizing immunochemicals on a particulate carrier, effective as diagnostic reagents for immunological tests for detecting or measuring a component in human or animal body fluids or for labeling cells.
In immunologically detecting or quantitatively analyzing either an antigen or antibody using the reaction between the antigen and antibody, it is an important method in immunological tests in clinical laboratories to immobilize a substance reactive with a second substance to be detected on a particulate carrier and to conduct a high sensitivity measurement by utilizing the phenomenon of agglutination which the above immobilized substance-carrier combination particles undergo in the presence of the substance to be detected. Also used widely in clinical laboratory tests is the method of immobilizing a substance to be detected on a particulate carrier, then utilizing the fact that agglutination of particles immobilizing the substance to be detected due to the presence of an antigen or antibody which specifically reacts with the substance to be detected is inhibited by the presence of the substance to be measured in the body fluids, and thereby detecting or quantitatively analyzing the substance to be detected. Moreover, the method of immobilizing a suhstance, which selectively binds to specific cells, on a particulate carrier and labeling the cells by determining whether or not the particles bind to the cells, has frequently been used for immunological testing.
Such immobilized immunochemicala-particulate carrier combinations are referred to as "immunoparticles".
As a particulate carrier as a part of a diagnostic reagent for immunological tests using such immunoparticles for agglutination reaction, substances used include red corpuscles of mammals or birds, particles of inorganic substances such as kaolin and carbon, and latex of organic high polymers such as natural rubber latex and polystyrene latex. Of these, red corpuscles can immobilize many kinds of antigens and antibodies and the applicable range thereof is the broadest. However, red corpuscles involve problems such that they differ in quality depending upon the individual animals from which they are drawn, are difficult to store because of insufficient stability and are sometimes non-specifically agglutinated by human serum.
It is polystyrene particles that are used most widely as non-organism originating carrier particles. Polystyrene is stable and because it is a synthetic polymer, the quality can be controlled. Because polystyrene is hydrophobic and has a property of adsorbing various proteins, immobilization of an antigen or antibody on polystyrene usually is carried out by physical adsorption. When an antigen or antibody is immobilized by physical adsorption, an equilibrium may occur between the immobilized antigen (or antibody) and a free antigen (or antibody) and result in a competitive reaction which takes place between the antigen (or antibody) immobilized on particles and the free antigen (or antibody) toward a corresponding antibody (or antigen) which is an objective substance of the measurement. This competitive reaction works to inhibit agglutination. As a result, there occur insufficient sensitivity and stability in many instances. Moreover, as a matter of course, substances difficult to be physically adsorbed to polystyrene cannot be immobilized by this method. Because of these problems, the practical application of polystyrene particles is limited as compared with red corpuscles as a carrier.
With a view to solving the aforesaid problems, it has recently been proposed to use other reagents prepared by bonding an antigen or antibody to a carrier by covalent bonding, such as reagents (see DT 2,649,218) prepared by bonding human chorionic gonadtropin to a styrene - methacrylic acid copolymer latex by using carbodiimide; reagents (see Japanese Patent Publication No. 12966/1978) comprising particles 0.01 to 0.9 microns in diameter prepared by condensing human chorionic gonadtropin, human serum albumin or denatured .gamma.-globulin, via amide bond and using carbodiimide as a condensing agent, to various latices such as carboxylated styrene -butadiene copolymer, carboxylated polystyrene, carboxylated polystyrene having an amino group, acrylic acid polymer, acrylonitrile polymer, methacrylic acid polymer, acrylonitrile - butadiene styrene terpolymer, polyvinyl acetate acrylate, polyvinyl pyridine and vinyl chloride - acrylate copolymer; reagents [see "The Japanese Journal of Clinical Pathology", 27, Supplementary Edition, page 522 (1978)]prepared by copolymerizing methacrylic acid, 2-hydroxyethyl methacrylate and methyl methacrylate and bonding treponema antigen to a latex of the resulting copolymer containing hydroxyl group and carboxyl group by the cyanogen bromide or carbodiimide method; and reagents (see Japanese Patent Laid Open No. 110118/1980) prepared by coating polystyrene particles as a core with styrene - glycidyl methacrylate copolymer and reacting human chorionic gonadtropin or insulin with an epoxy group in the latex thereby bonding it to the latex. Many of these prior art methods use carbodiimide for bonding immunochemicals to carrier particles. But the use of carbodiimide would cause an inter- or intra-molecular condensation reaction of the immunochemicals, but in this case it is difficult to obtain the reproducibility of reaction between hydroxyl group-containing polymer and cyanogen bromide. As a result, the immunoactivity of particles with immunochemicals immobilized thereon lacks in reproducibility. As compared with these immunochemicals immobilizing methods, the method of reacting proteins or polypeptides with an epoxy group introduced in polymer causes less deterioration of immunoactivity and is superior in the reproducibility of the reaction. In the above-mentioned prior art using an epoxy group, however, proteins tend to be adsorbed non-specifically because on the surfaces of the polymer particles there exists a portion originating from styrene. Generally, in human or animal body fluids there are contained various kinds of proteins, and particularly in blood plasma and serum there are contained those proteins at high concentrations. When protein is adsorbed onto carrier particles from the test body fluids, it may interfere with the objective antigen-antibody reaction and cause a reduction in the selectivity or sensitivity of the agglutination reaction.